Global Dynamic Conformational Changes in the Suppressor Domain of IP3 Receptor by Stepwise Binding of the Two Lobes of Calmodulin

Abstract

The roles of calmodulin (CaM) has been a key point of controversy in the regulation of inositol-1,4,5-trisphosphate receptor (IP3R). Current views differ in terms of the involvement of CaM as calcium sensors and calcium's functions in the process. To help resolve these issues, we studied the interaction between CaM and the suppressor domain of IP3R, a key allosteric regulatory domain, in the absence and presence of calcium. Through NMR binding experiments, we observed dramatic peak disappearances of the suppressor domain upon interaction with apo-CaM. These data indicated that apo-CaM induces large-scale dynamic conformational changes in the suppressor domain, most probably involving partial unfolding and sub-domain rearrangement. Resonance assignments of CaM surprisingly revealed that its C-lobe alone can cause these changes. Subsequent NMR binding experiments showed that calcium allows the free N-lobe to additionally bind to the suppressor domain, which induces extra conformational changes in both of the proteins. Our data also suggest that the extra changes in the suppressor domain are secondary to those in calmodulin. Based on these results, we propose that apo-CaM, through its C-lobe, can prime the allosteric regulation by partially unfolding the suppressor domain, which could be propagated to distant sites to open inhibitory calcium binding sites. Calcium then could bind to the CaM's N-lobe and the inhibitory binding sites in IP3R, eliciting additional conformational changes and actual inhibition of IP3R. We believe that our results reconcile previous allosteric models and provide new insights in the mechanism of calcium/CaM-mediated regulation of IP3R.